Use of specific T2R taste receptors to identify compounds that block bitter taste

Abstract

Assays for identifying compounds that modulate, preferably inhibit bitter taste associated with the activation of hT2R4, hT2R44 and/or hT2R61 are provided. The compounds identified according to these assays should modulate, e.g., inhibit bitter taste associated with bitter tasting compounds including quinine, 6-nitrosaccharin, saccharin and/or denatonium. These compounds are useful additives for foods, beverages or medicinal preparations having a bitter taste.

1. An assay method for identifying a compound which modulates hT2R61 associated bitter taste comprising:
 (i) screening a compound for its effect on the activation of an hT2R61 taste receptor polypeptide that is at least 95% identical to the hT2R61 polypeptide contained in SEQ ID NO:5; and 
 (ii) determining whether said compound modulates hT2R61 associated bitter taste based on its effect on the activation of said taste receptor polypeptide by 6-nitrosaccharin or saccharin. 
 
     
     
       2. The assay of  claim 1  wherein said hT2R61 taste receptor polypeptide is at least 96% identical to the polypeptide contained in SEQ ID NO:5. 
     
     
       3. The assay of  claim 1  wherein the hT2R61 taste receptor polypeptide is at least 97% identical to the polypeptide contained in SEQ ID NO:5. 
     
     
       4. The assay of  claim 1  wherein the hT2R61 taste receptor polypeptide is at least 98% identical to the polypeptide contained in SEQ ID NO:5. 
     
     
       5. The assay of  claim 1  wherein said hT2R61 taste receptor polypeptide is at least 99% identical to the polypeptide contained SEQ ID NO:5. 
     
     
       6. The assay of  claim 1  wherein said hT2R61 taste receptor polypeptide has the sequence contained in SEQ ID NO:5. 
     
     
       7. The assay of  claim 1  wherein a compound which is identified as modulating the activation of said hT2R61 taste receptor polypeptide by 6-nitrosaccharin or saccharin is further tested in a taste test. 
     
     
       8. The assay of  claim 1  wherein said hT2R61 taste receptor polypeptide is expressed on a cell membrane. 
     
     
       9. The assay of  claim 8  wherein the cell membrane is isolated. 
     
     
       10. The assay of  claim 8  wherein the cell membrane is from an insect or mammalian cell. 
     
     
       11. The assay of  claim 1  wherein the hT2R61 polypeptide is expressed on a cell selected from a HEK cell, COS cell, BHK cell and a CHO cell. 
     
     
       12. The assay of  claim 1  which screens the effect of said compound on hT2R61 associated taste by detecting if there is a change in intracellular ion concentration in the presence of the compound after saccharin or 6-nitrosaccharin induced activation. 
     
     
       13. The assay of  claim 12  wherein the ion is calcium. 
     
     
       14. The assay of  claim 12  wherein the ion is sodium. 
     
     
       15. The assay of  claim 1  which screens the effect of said compound on membrane potential in the presence of said compound after 6-nitrosaccharin or saccharin induced activation of said hT2R61 taste receptor polypeptide. 
     
     
       16. The assay of  claim 1  wherein the assay selects for a compound that blocks the interaction of said hT2R61 taste receptor polypeptide with a 6-nitrosaccharin or saccharin containing compound. 
     
     
       17. The assay of  claim 1  wherein the hT2R61 taste receptor polypeptide is encoded by a gene which is operably linked to a gene encoding a polypeptide sequence that facilitates transport of the hT2R61 polypeptide across a cell membrane. 
     
     
       18. The assay of  claim 1  wherein the hT2R61 polypeptide is expressed by a HEK-293 cell line. 
     
     
       19. The assay of  claim 1  wherein the assay detects the effect of said compound on saccharin or 6-nitrosaccharin induced hT2R61 activation based on its effect on at least one of intracellular cAMP, cGMP and IP3 concentration. 
     
     
       20. The assay of  claim 18  which detects the effect of said compound on intracellular calcium using a calcium specific reporter molecule. 
     
     
       21. The assay of  claim 20  wherein the reporter is a fluorescent dye specific for calcium. 
     
     
       22. The assay of  claim 21  wherein the dye is selected from Fura-2, Fluo-3 and Fluo-4. 
     
     
       23. The assay of  claim 1  wherein the hT2R61 polypeptide is attached to a reporter molecule. 
     
     
       24. The assay of  claim 23  wherein said reporter is a green fluorescent protein. 
     
     
       25. The assay of  claim 1  wherein the hT2R61 polypeptide is attached or comprised in a bilayer membrane, solid phase, or a vesicle. 
     
     
       26. The assay of  claim 1  which detects the effect of said compound on the interaction of said hT2R61 polypeptide with gustducin or transducin. 
     
     
       27. The assay of  claim 1  wherein the assay is a fluorescence polarization assay. 
     
     
       28. The assay of  claim 1  which is a high throughput assay that screens the effect of different compounds on the activation of said hT2R61 polypeptide by saccharin or 6-nitrosaccharin. 
     
     
       29. The assay of  claim 1  wherein said hT2R61 polypeptide is expressed by a cell line that stably expresses said polypeptide. 
     
     
       30. The assay of  claim 1  wherein said hT2R61 polypeptide is expressed by a cell line that transiently expresses said polypeptide.